1. Field of the Invention
The present invention concerns a modified microorganism and its use for the preparation of 1,2-propanediol and/or acetol.
2. Description of Related Art
1,2-propanediol or propylene glycol, a C3 dialcohol, is a widely-used chemical. It is a component of unsaturated polyester resins, liquid detergents, coolants, anti-freeze and de-icing fluids for aircraft. Propylene glycol has been increasingly used since 1993-1994 as a replacement for ethylene derivatives, which are recognised as being more toxic than propylene derivatives.
1,2-propanediol is currently produced by chemical means using a propylene oxide hydration process that consumes large amounts of water. Propylene oxide can be produced by either of two processes, one using epichlorhydrin, and the other hydroperoxide. Both routes use highly toxic substances. In addition, the hydroperoxide route generates by-products such as tert-butanol and 1-phenyl ethanol. For the production of propylene to be profitable, a use must be found for these by-products. The chemical route generally produces racemic 1,2-propanediol, whereas each of the two stereoisomers (R)1,2-propanediol and (S)1,2-propanediol are of interest for certain applications (e.g. chiral starting materials for specialty chemicals and pharmaceutical products).
Acetol or hydroxyacetone (1-hydroxy-2-propanone) is a C3 keto alcohol. This product is used in vat dyeing process in the textile industry as a reducing agent. It can advantageously replace traditional sulphur containing reducing agents in order to reduce the sulphur content in wastewater, harmful for the environment. Acetol is also a starting material for the chemical industry, used for example to make polyols or heterocyclic molecules. It possesses also interesting chelating and solvent properties.
Acetol is currently produced mainly by catalytic oxidation or dehydration of 1,2-propanediol. New processes starting from renewable feedstocks like glycerol are now proposed (see DE4128692 and WO 2005/095536). Currently, the production cost of acetol by chemical processes reduces its industrial applications and markets.
The disadvantages of the chemical processes for the production of 1,2-propanediol and/or acetol make biological synthesis an attractive alternative. Two routes have been characterized for the natural production of these products from sugars by microorganisms.
In the first route 6-deoxy sugars (e.g. L-rhamnose or L-fucose) are cleaved into dihydroxyacetone phosphate and (S)-lactaldehyde, which can be further reduced to (S)-1,2-propanediol (Badia et al, 1985). This route is functional in E. coli, but can not yield an economically feasible process due to the elevated cost of the deoxyhexoses.
The second route is the metabolism of common sugars (e.g. glucose or xylose) through the glycolysis pathway followed by the methylglyoxal pathway. Dihydroxyacetone phosphate is converted to methylglyoxal that can be reduced either to lactaldehyde or to acetol. These two compounds can then undergo a second reduction reaction yielding 1,2-propanediol. This route is used by natural producers of (R)-1,2-propanediol, such as Clostridium sphenoides and Thermoanaerobacter thermosaccharolyticum. Clostridium sphenoides has been used to produce 1,2-propanediol at a titer of 1.58 g/l under phosphate limited conditions (Tran Din and Gottschalk, 1985). Thermoanaerobacter thermosaccharolyticum has also been investigated for the production of 1,2-propanediol (Cameron and Cooney, 1986, Sanchez-Rivera et al, 1987). The best performances obtained were a titer of 9 g/l and a yield from glucose of 0.2 g/g. However, the improvement of the performances obtained with these organisms is likely to be limited due to the shortage of available genetic tools.
E. coli has the genetic capabilities to produce naturally 1,2-propanediol and acetol. The biosynthetic pathway to 1,2-propanediol starts from the glycolysis intermediate dihydroxyacetone phosphate. This metabolic intermediate can be converted to methylglyoxal by methylglyoxal synthase encoded by mgsA gene (Cooper, 1984, Tötemeyer et al, 1998). Methylglyoxal is an extremely toxic electrophile that can react with nucleophilic centres of macromolecules such as DNA, RNA and proteins. It can inhibit bacterial growth and cause cell death at very low concentrations (0.3 to 0.7 mM). For this reason, the existing routes for detoxification of methylglyoxal have been investigated (Ferguson et al, 1998). Three pathways have been identified in bacteria and specifically in E. coli:                 The first one is the gluthathione dependent glyoxalase I-II system (encoded by gloA and gloB genes) which converts methylglyoxal into D-lactate in two steps.        The second one is the glutathione independent glyoxalase III enzyme which catalyses the conversion of methylglyoxal into D-lactate.        The third system encompasses the degradation of methylglyoxal by methylglyoxal reductases.This last system is relevant for the production of 1,2-propanediol. Methylglyoxal is a C3 ketoaldehyde, bearing an aldehyde at C1 and a ketone at C2. Theses two positions can be reduced to alcohol, yielding respectively acetol (or hydroxyacetone), a non-chiral molecule and lactaldehyde, a chiral molecule which can exist in L- or D-form (see FIG. 1). These 3 molecules, acetol, L-lactaldehyde and D-lactaldehyde can be subsequently reduced at the other position to yield chiral 1,2-propanediol.        
The pathways preferentially used in E. coli are not clearly established at this time. A methylglyoxal reductase, using preferentially NADPH as co-factor, was purified and partially characterized in E. coli (Saikusa et al, 1987). The product of this reaction was shown to be lactaldehyde. Misra et al (1996) described the purification of two methylglyoxal reductase activities giving the same product acetol. One NADH dependent activity could be an alcohol dehydrogenase activity whereas the NADPH dependent activity could be a non-specific aldehyde reductase. Altaras and Cameron (1999) demonstrated that glycerol dehydrogenase (GldA) encoded by the gldA gene of E. coli is active in reducing methylglyoxal to (R)-lactaldehyde, and also in the conversion of acetol into 1,2-propanediol.
The gene yghZ was cloned from E. coli, expressed and the protein was characterized (Grant, 2003). It exhibited a high specific activity toward methylglyoxal with NADPH as a co-factor, but the product of the reaction was not characterized. When overexpressed, this gene conferred resistance to methylglyoxal toxicity.
Ko et al (2005) investigated systematically the 9 aldo-keto reducases of E. coli as candidates for the conversion of methylglyoxal into acetol. They showed that 4 purified enzymes, YafB, YqhE, YeaE and YghZ were able to convert methylglyoxal to acetol in the presence of NADPH. According to their studies, the methylglyoxal reductases YafB, YeaE and YghZ would be the most relevant for the metabolism of methylglyoxal in vivo in terms of detoxification. Di Luccio et al (2006) showed that the product of the gene ydjG of E. coli is active on methylglyoxal with NADH but the characterization of the product of the reaction was not done.
Several investigations for genetic modifications of E. coli in order to obtain a 1,2-propanediol producer using simple carbon sources have been done by the group of Cameron (Cameron et al, 1998, Altaras and Cameron, 1999, Altaras and Cameron, 2000) and the group of Bennett (Huang et al, 1999, Berrios-Rivera et al, 2003). These studies rely on the expression of one or several genes coding for enzymatic activities in the pathway from dihydroxyacetone phosphate to 1,2-propanediol. Cameron et al (1998) showed that the overexpression of either the gene coding for rat lens aldose reductase or the gldA gene resulted in the production of less than 0.2 g/l 1,2-propanediol. Improvement of this titer can be obtained by co-expressing two E. coli genes, mgsA and gldA. With this combination, a titer of 0.7 g/l 1,2-propanediol can be obtained (Altaras and Cameron, 1999). Further improvement in titers and yield were obtained when expressing a complete 1,2-propanediol pathway in E. coli (Altaras and Cameron, 2000). Three genes, mgsA, gldA and fucO, have been overexpressed in a strain lacking the gene coding for lactate dehydrogenase (ldhA). With this combination, the best results obtained by the group of Cameron are production of 1.4 g/l 1,2-propanediol in anaerobic flask culture with a yield of 0.2 g/g of glucose consumed. When extrapolated in anaerobic fed-batch fermenter, the production was 4.5 g/l of 1,2-propanediol with a yield of 0.19 g/g from glucose. Results obtained with the same approach but with lower titers and yields are also described in U.S. Pat. No. 6,087,140, U.S. Pat. No. 6,303,352 and WO 98/37204. The group of Bennett also used an E. coli host strain lacking ldhA for the overexpression of the mgs gene from Clostridium acetobutylicum and the gldA gene from E. coli. Flask cultures under anaerobic conditions gave a titer of 1.3 g/l and a yield of 0.12 g/g whereas microaerobic cultures gave a titer of 1.4 g/l with a yield of 0.13 g/g.
At this stage, all these results are not better than those obtained with the species T thermosaccharolyticum.
Up to now, the use of endogeneous activities from microorganisms, and in particular from E. coli, converting methylglyoxal to acetol has not been described.